biochem exam 2

1. what are enzymes?

2. why are they important to life?

1. enzymes = proteins biological catalysts

2. they efficiently and selectively catalyze chemical reactions

4 benefits of catalysts for biochemistry

1. increase reaction rates

2. allow milder reaction environments (bodily conditions)

3. greater reaction specificity for substrates and products

4. capacity for regulation

can enzymes catalyze L and D isomers of their substrate?

No, enzymes are isomer/stereo-specific

4 mechanisms that allow regulation of enzymes

1. allostery

2. covalent modification

3. assembly into macromolecular complexes

4. feedback inhibition

1. allostery-

2. covalent modification-

3. feedback inhibition

1. allostery = binding of an inhibitor/activator at one site regulates activity at the substrate-binding site

2. phosphorylation, acetylation, removal of inhibitors, etc…

3. a product in a pathway regulates an earlier catalytic enzyme in the same pathway

1. apoenzyme (apoprotein)

2. holoenzyme

1. apoenzyme (apoprotein) = without its cofactor, nonfunctional

2. holoenzyme = cofactor bound, functional

1. active/catalytic site

2. binding site

3. cofactor

4. coenzyme

5. difference b/t cofactor and allosteric modulator?

1. enzyme site where reaction happens : substrate → product

2. the main part of the active site that binds substrate

3. cofactor = nonprotein helper mC/metal ion needed for enzyme to catalyze a reaction.. binds the active site

4. coenzyme = organic cofactor usually made of vitamins. may bind transiently or tightly (prosthetic groups). required as carriers of specific atoms or functional groups

5. cofactor binds active site

why do we need to intake vitamins?

used as cofactors for our enzymes. we will get sick if we don’t have them

differentiate b/t (under what conditions):

1. dG,

2. dG-zero,

3. dG’-zero

4. dG-double-dagger

1. for what reaction are we using them?

1. dG = free energy change under any conditions

2. dG-zero = standard free energy change

   1. 298K, 1M, 1 atm, etc…

3. dG’-zero = biochemical standard free-energy change

   1. biological conditions = 1M pH7

4. dG-double-dagger =activation energy

1. any reaction substrate(s) → product(s)

DG’0 v. dG-double-dagger… what info do these give us about a S→P reaction?

for a given reaction S→P":

1. dG’-zero = how likely that a reaction will happen

   1. thermodynamics

2. dG-double dagger = how fast it will happen

   1. kinetics

what happens at transition state?

50% chance of product or substrate

General free energy Reaction Graph:

1. x and y axis

2. where are the substrate and product ground states

3. what is double dagger

4. 2 dG-double daggers

5. standard free energy change?

1. s

2. 
   

3. 
   

1. will the product be made/why?

2. how fast will it take for the product to be made/why? (large DG-double dag)

3. what about if P→S?

1. yes bc -dG (energetically favorable) b/t S → P

2. a long time bc there’s a large activation energy

3. less likely bc even more activation energy to overcome

   1. not energetically favored

1. exergonic v. endergonic?

2. what if dG = 0?

1. exergonic = -dG, energy released, spontaneous/favorable

2. endergonic - +dG, energy absorbed, not spontaneous/favorable

3. equilibrium, nothing favorable

for reaction S→P:

1. what is k?

2. how is it related to dG-double dagger?

3. how is it related to reaction speed?

1. k = rate constant

2. big dG-double dagger = small k = slow reaction

1. uncatalyzed reaction

2. catalyzeddreaction

3. reaction intermediate

4. rate limiting step

5. do enzymes catalyze both forward and reverse reactions?

1. S ← k → P

2. S+E ←k1→ ES ←k2→ EP ←k3→ P+E

3. ES and EP are detectable reaction intermediates

4. step with the highest barrier energy/lowest rate constant k

5. yes

how do enzymes affect equilibrium?

1. they don’t set equilibrium they simply get you there faster

1. why enzymes needed?

2. explain how enzyme accelerate reaction

   1. cat v uncut

3. do enzymes affect dG, dG’-zero?

1. if dG-double dagger (activation energy) high, the rxn won’t happen

2. enzymes lower dG-double dagger

   1. dG-double dagger (cat) < dG-double dagger (uncat

3. NO, do nothing to difference in free energy b/t the substrate and product as that is chemical differences. ONLY reduces activation energy to get to transition state

1. 3 steps enzyme catalysis

2. how do enzymes bind to substrate?

3. what is DG-B?

   1. what is it the result of?

   2. what is the effect?

1. initially binds unstressed substrate → when fully bound, substrate at transition state → substrate forms products

2. enzyme active site is complementary to/optimized for transition state (stressed state)

3. binding energy = difference b/t activation energies (dG-double dagger) of the uncatalyzed vs. catalyzed reaction

   1. many weak interactions/bonds b/t enzyme and substrate

   2. activation E reduced

1. 1 main but 4 things contribute to a high activation energy (dG-double dagger)?

2. how do enzymes overcome

1. entropy reduction (entropically unfavored)

   1. desolved substrates

      1. enzyme gets water out of the way

   2. flexible/distorted substrates

      1. binding causes substrate conformation change towards transition state, holds it rigid

   3. catalytic functional groups on E must be properly aligned

      1. induced fit = enzyme binding causes enzyme to change conformation

1. main reason activation E high

   1. uncatalyzed bimolecular reactions

   2. uncatalyzed unimolecular reactions

1. why catalyzed reactions different?

1. entropy reduction is what makes activation E high:

   1. both are entropically unfavored

      1. two free reactants → single restricted transition state

      2. flexible reactant → rigid transition state

   2. catalyzed reactions = favorable

      1. entropy cost is paid during binding. puts subtrate into rigid ES complex

      2. rigid reactant complex → rigid transition state = entropically OK

why protein structure important for enzymatic function?

tertiary and quaternary protein creates cradle for the active site

1. is dG-B the only way enzymes catalyze?

2. 3 other ways

1. no, some enzymes do more than stabilize transition states

2. acid-base catalysis

3. covalent catalysis

4. metal/ion catalysis

acid-base catalysis:

1. what is it

2. why is it needed

3. specific acid-base catalysis v. general acid-base catalysis

4. what group is the acid/base in an enzyme? / where

   1. which is most popular and why?

1. when acids/bases neutralize charges by donating or abstracting proton

2. charged intermediates aren’t stable and can decay back to reactants

1. specific = H+ donation/abstraction is carried out by water

2. general = H+ donation/abstraction carried out by enzyme

1. ionizable AA R groups/side chains act as acids/bases in the active site

   1. histidine bc it has aKa closest to physiological pH

covalent catalysis:

1. what is it

2. result?

3. why does this catalysis happen?

1. a covalent E-S complex forms that helps drive the reaction forward

2. a new reaction pathway than the uncatalyzed reaction

3. steps of forming and breaking covalent bond are faster (lower dG-double dagger) than uncatalyzed reaction

metal ion catalysis:

1. metals normally are enzyme ____

2. 3 ways it can help catalyze substrate

1. cofactors

2. 3 ways

   1. can orient substrates

   2. contribute to binding E (stabilized transition state

   3. can donate/absorb protons = change redox state

V0:

1. what is it

2. equation

3. why focus on V0

4. what determines V0? why?

1. rate at which product is being formed at time 0

2. delta [P] / delta time

3. initial useful bc velocity decreases over time as substrate drops and products form causes reverse reaction to start

4. V0 proportional to [S], but only at lower [S] concentrations!

   1. as [S] rises, V0 approaches Vmax bc enzymes get saturated. when they saturate, adding substrate won’t increase velocity

enzyme kinetics plot:

1. x and y axis, variables

2. Michaelis menton equation

3. units for x and y

1. x axis = [S], y = [V0]. Km is [S] when V0 =½ Vmax

   1. ½ Vmax like theta, Km like ki

2. V0 = Vmax x [S] / Km + [S]

3. V0 = uM/min, [S] = mM

3 assumptions for V0 for S+e - ES - P+E

1. steady state assumption = in the initial reaction, delta [Etotal] / delta time = 0

   1. in the initial reaction, [ES] is constant. delta [ES]/delta t = 0

   2. [E] constant bc enzyme not being used up in reaction

2. second step, k2, is rate limiting. k2 determines V

   1. no k-2 bc no [P] in the beginning

3. at saturation, Vmax, [ES] = Etotal]

Michaelis Vmax variation V0 = (k2)(Etotal)[S] / Km+[S]:

1. 2 reasons why

2. Vmax = ?

3. Vmax is what?

1. 2 reaons

   1. V0 approaches Vmax as [S] increases, but only until enzyme saturated, (when [ES] = [Etotal] )

2. Vmax = k2 x [Etotal]

3. at Vmax, enzyme saturated, adding [S] won’t increase rate

Km the Michaelis constant:

1. equation

2. what does it represent

3. when is the equation true

4. Km relationship to biological substrate concentration? why?

5. small Km = ?, big Km = ?

1. Km = (k2 +k-1) / k1

2. Km = [S] at which V0 is ½ Vmax.

   1. indirect measure of ES binding/affinity of substrate for an enzyme/how well substrate binds enzyme

3. when k2 is the rate limiting step

4. sits around Km (1/2 Vmax) so it can be up or down regulated

5. its like Kd, small Km means tight binding (high affinity), large = weak binding (low affinity)

double reciprocal kinetics plot (line weaver burke plot):

1. why?

2. y intercept

3. x intercept

4. slope

5. equation

1. turns exponential plot into linear plot where its easy to find variables

2. y intercept = 1/Vmax

3. x intercept = -1/Km, but 1/Km if just looking at it to find Km

4. slope = Km/Vmax

5. equation: (1/V0) = (Km/Vmax) (1/[S]) + (1/Vmax)

   1. y = mx + b

1. What happens when to MM when [S] = Km

2. is Km an affinity constant

3. what kind of reactions

1. which substrate does hexokinase process faster? which does hexokinase have higher affinity for?

1. V0 = Vmax / 2

2. no, indirect measure of affinity for SIMPLE enzyme reactions (E+S-ES-E+P) bc acts as dissociation constant for ES

   1. more steps = less reliable

3. glucose for both

1. 2 differences between kinetics plot at low [S] v high [S]?

2. what is [S] and V0 at biological conditions? why?

1. low [S]

   1. linear relationship b/t [S] and V0, Michaelis Minton equation

   2. more [E] than [ES]

2. high [S]

   1. V0 approaches Vmax

   2. [ES] approaches [Etotal]

Km (MM) v. Kd (ligand binding w/ theta)

1. Km = measures the “affinity” of an enzyme to its substrate/ligand, even though its more a measurement of velocity

   1. V0 = Vmax + [S] / Km + [S]

2. Kd = measures how “tight” and enzyme binds to substrate/ligand

   1. theta = [L] / [L] + Kd

kcat:

1. what is it

2. specifically

3. turnover #

4. units

5. what is kcat for reaction in image

6. what is Vmax equation adjustment for the reaction in the image

7. when do you want a small kcat (slow enzyme)

1. kcat = limiting rate of an enzyme catalyzed reaction at saturation

   1. rate limiting step AT SATURATION

2. kcat = “turnover number” = # mCs substrate converted to product by 1 enzyme per second

3. unit = sec^-1

4. Vmax = k2[ET] = kcat[ET] if k2 is rate limiting step

   1. kcat = k3 if k3 is rate limiting step

5. for balance. don’t want 1 enzyme using all of that substrate, also don’t want to produce too much product

specificity constant:

1. what is it equation

2. how is it useful

3. what does it tell you

4. new V0 equation

   1. when

1. kcat/Km (M-1sec-1)

2. comparing how good two enzymes or an enzyme working on 2 diff substrates are

3. catalytic efficiency = how efficiently an enzyme catalyzes the reaction in each encounter b/t E and S

4. V0 = (kcat/Km) x [Et] x [S]

Catalytic perfection:

1. what is it based on

2. when does it happen

1. diffusion limit of E an S in the cell = 10^8 - 10^9

   1. how fast 2 things in the cell can come together/diffuse together

2. specificity constant (catalytic efficiency) reaches the diffusion limit, the enzyme is so efficient that it catalysis happens every time E meets S = catalytic perfection

enzymes that have 2 enzymes:

1. ternary complex

   1. random order

   2. ordered

2. no turnary complex

enzymes that have 2 enzymes:

1. ternary complex = when enzyme is bound to more than 1 substrate at the same time

   1. random order = enzyme can bind substrate 1 or 2 and then the other in any order

   2. ordered = enzyme has to bind 1 substrate before the other

2. ping pong mechanism = no ternary complex

   1. enzyme is modified by first substrate, producing a product that we don’t care about and the reaction continues to get ultimate product, ultimately enzyme unchanged

1. feedback inhibition

first enzyme in a pathway allosterically down-regulated by the product of the pathway

pre-steady state v. steady state amounts: which applies for Michaelis menten

1. substrate

2. Enzyme

3. ES

4. Product

1. pre steady state

   1. substrate = very high

   2. Enzyme = high

   3. ES = low

   4. Product = low

2. steady state = when MM is used

   1. E and ES both relatively constant

1. top hat

2. how to find Vmax and Km from data

3. from LB plot (image in answer card)

1. Vmax = 150 (highest V and its plateuing)

   1. good estimate, actual may be 151

2. Km = 8

   1. [S] at ½ Vmax

1. Vmax =

   1. 1/y intercept

   2. slope = Km/Vmax, Vmax = “run”

2. Km =

   1. 1/x intercept if you ignore the negatives. technically its -1/Km = x intercept, but since x intercept is neg, just ignore it

   2. slope = Km/Vmax, Km = “rise”

1. left V0 = 1/5 uM/min, left Km = ¼ mM

2. right V0 = 1/200 uM/min, right Km = ½ mM

what else can you get from a LB plot/how?

1. k2 kcat and Vmax

2. specificity constant?

1. for simple reactions, kcat = k2

2. Vmax = k2 [ES], but ES = Et when maxed out, so Vmax = k2[Et]

3. k2 = kcat, so Vmax = kcat[Et]

top hat

c

some more regulations of enzymes

1. homotropic allostery

2. heterotropic allostery

3. phophorylation

4. suicide inhibitor

5. cleavage

1. homotropic allostery = regulatory mC (effector) is the substrate

2. heterotropic allostery = regulatory mC is not substrate

3. phophorylation =

4. suicide inhibitor = mimics substrate permanently covalently binds active sight and stops activity

5. cleavage = cleaving an inactive protein to an active state

cleavage:

1. what happens

2. why

3. nomenclature proteins v enzymes

1. inactive precursors converted to active enzymes by enzymatic (specific cleavage)

2. many enzymes produced in an inactive form called a zymogen = proenzyme

3. nomenclature

   1. pro at the beginning is for inactive uncleaved proteins

   2. “gen” at end for inactive uncleaved enzymes (zymogens) = chymotrypsinogen is inactive form of chymotrypsin

covalent modifications:

1. what are they

2. what do they do

3. how

4. what does it result in

1. covalent modification = variety of permanent, not transient modifications possible, can only be changed by enzyme

2. affect structure and function

3. phosphorylation can activate/deactivate enzyme, enzyme is phosphorylated/dephosphorylated by enzymes

4. many things

   1. enzyme on/off, change localization, change substrate preference, degrade, etc…

phosphorylation:

1. what is it

2. who does it/who removes it

   1. how specific

1. covalent attachment of phosphate

2. kinases attach phosphate

   1. specific = recognizes motif (specific AA sequence), usually serine threonine, tyrosine

3. phosphatase removes phosphate

   1. less specific

glycogen phosphorylase:

1. what does it do

2. 2 forms

3. how switch b/t forms

1. turns glucose into glycogen,

2. switches b/t active a form and less active b form via phosphorylation

3. phosphorylation of 2 Serines convert b to a

   1. phosphorylase kinase coverts b to a = activates

   2. proteins phosphatase 1 converts a to b = inhibits

enzyme inhibitors:

1. what do they do

   1. how

2. what do folic acid metabolism inhibitors do

3. prostaglandin synthestase inhibitor

4. aromatic amino acid synthesis inhibitor

1. reduce enzyme activity

   1. mimics substrate

   2. mimic transition structure

   3. reducing turnover number kcat

2. prevent cancer by inhibit building block of DNA to stop proliferating

enzyme inhibitors:

1. reversible v irreversible

2. how regulated metabolic processes

3. 3 reversible 1 irreversible

4. how do they affect MM / LB equations

1. reversible = not permanent, can bind and unbind

2. feedback inhibition

3. 3 reversible

   1. competitive = mimics substrate binds in active site

   2. uncompetitive = not directly competing for active site, but modulates ES complex

   3. mixed

1. “apparent” constants ; can impact Vmax and Kmapp in different ways

   1. V0 = Vmaxapp x [S] / Kmapp + [S]

competitive inhibition:

1. what is it

2. how affect Vmax and Km

3. what happens if add more S while [I] is constant?

4. affect on MM

5. affinity calculation Ki

1. reversible inhibitor competes directly w/ substrate for enzymes’s binding site

2. doesn’t affect Vmax, affects apparent Km (alpha)

3. more S = more product if overcome inhibition

4. V0 = Vmax[S] / [S] + alpha x Km

5. “affinity” = Ki = [E][I] / [E]

1. competitive inhibitor affect on Km:

   1. affectt of more competitive inhibition on Km

   2. what is alpha of no inhibition?

   3. alpha equation

2. what about Vmax? meaning?

3. how do you reverse effect of competitive inhibitor?

1. affect on Km

   1. as alpha increases, Km increases

      1. makes Km appear larger/ [S] more dilute

      2. Kmapp = alphaxKm > Km, when comp inhibitor present

   2. alpha = 1 when no inhibiiton. alpha > 1 = inhibitor

   3. alpha = 1 + [I]/Km

2. competitive inhibitor no affect on Vmax, if you add [S] eventually you will reach Vmax

   1. BUT need higher [S] to reach Vmax as alpha increases

3. reverse affect by adding [S]

1. competitive inhibitor affect on LB

   1. 1/Vmax?

   2. 1/Km?

2. on MM plot?

1. competitive inhibitor = turns counter clockwise (more vertical) as alpha rises

   1. no change in 1/Vmax

   2. adding inhibitor → higher Kmapp

2. MM plot

uncompetitive inhibition:

1. what is it

2. Ki équation

3. MM modification

4. how affect Vmax and Km

5. 3 rules

   1. high [S] values

   2. kcat

   3. increasing [S]?

1. inhibitor binds to ES-complex, but not to the free enzyme

   1. not directly competing w/ substrate for active site

2. Ki = ES][I]/[ESI

3. V0 = Vmax)[S]/ alpha' x Km x [S]

4. affects Vmax, affects Km

   1. more inhibitor = lower Vmax

   2. more inhibitor = smaller Km

5. 3 rules

   1. at high values of [S], V0 approaches Vmax/alpha’

   2. catalytic function kcat reduced by not substrate binding

   3. affect of uncompetitive inhibitor can’t be reversed by increasing [S]

1. uncompetitive inhibitor affect on LB

   1. 1/Vmax?

   2. 1/Km?

2. on MM plot?

1. higher alpha’ (more inhibitor) = raises line vertically

   1. smaller inhibitor = smaller Vmax

   2. more inhibitor = smaller Km

2. Km and Vmax drop

mixed inhibition:

1. what is it

2. Ki équation

3. MM modification

4. how affect Vmax and Km

5. 3 rules

   1. high [S] values

   2. kcat

   3. increasing [S]?

1. reversible inhibitor binding to E and ES at a site distinct from substrate

2. both Ki (competitive) and Ki’ (uncompetitive)

3. V0 = Vmax[S]/a’[S] + aKm

4. affects Vmax and Km

   1. higher inhibitor = Vmaxapp gets smaller, larger Km (depends on a/a’)

      1. larger a = larger Km

      2. smaller a = smaller Km

1. what is rate constant k

2. higher or lower speed

3. 4 rate constants for standard enzyme catalyzed reaction at any given time:

   1. E + S ←> ES ←> E + P

4. which 2 rate constants indicate the V (reaction rate)?

   1. which do we use, why?

5. In a typical enzyme-catalyzed reaction, when & why is the rate constant k -2 negligible?

Pearson

1. indicator of reaction rate efficiency under set conditions

2. higher k = faster, k = 0 means no reaction

3. 4 k’s

   1. k1 = rate constant for E+S → ES

   2. k-1 = rate constant for E+ S ← ES

   3. k2 = rate constant for ES → E+P

   4. k-2= rate constant for ES ←E + P

4. the k that affects the products, k2 and k-2

   1. k2 if we consider early reaction conditions when there is no product present

5. start of a reaction where S is highest, p is lowest, and reverse reaction unlikely

1. 3 rate constants for E+S ←> ES ←> E+ P at initial stages?

2. why no k-2?

3. what is the V for this moment, and what does the equation^ change to?

4. rate limiting step?

Pearson

1. k1, k-1, k2

2. no products present, [P] = 0

3. V0 = enzyme catalyzed reaction at t=0

   1. E+S ←> ES -> E+ P, no k-2

4. rate limiting step = slow step that dictates reaction rate = k2

1. [St] = ?

2. why?

Pearson

1. [S] bc normally [St] = [S] bc [S] > > > [E] and [ES] at V0

1. induced fit

2. general acid base catalysis

3. specific acid base catalysis

1. enzyme changes conformation upon substrate binding to help catalyze

2. general = mCs other than water, usually AA r groups donate/accept H+

3. specific = catalysis donating/accepting H+ carried out by water itself

for general enzyme catalyzed reaction:

1. equation

2. rate limiting step

   1. because it is the sloweststep/lowest k2

3. why no k-2

4. [E]t =E + ES

5. [S]t = S + ES

1. E + S - ES - E + P

2. rate limiting step = ES - E + P

   1. no P

3. why no k-2

4. [E]t =E + ES

5. [S]t = S + ES

what type of inhibitor?

what type of inhibitor?

competitive v. uncompetitive v. mixed inhibitors:

1. what do they do

1. competitive = compete w substrate in substrate binding site

   1. doesn’t change V max

2. uncompetitive = doesn’t compete in binding site but binds ES complex

   1. no matter how much substrate you add you can’t overcome it

3. mixed = not competitive, don’t just bind ES complex

   1. can bind ES complex and to the Enzyme w/o substrate

   2. cannot overcome with additional substrate

   3. affects both Km and Max

1. irreversible inhibitor:

2. suicide inhibitor

3. how medically relevant?

1. modify or destroy a catalytic functional group of an active site by covalent or extremely tight non covalent modification

   1. potent poisons

2. type of irreversible inhibitor that binds like a substrate and is then acted upon by enzyme

   1. at some point during catalysis converted into an irreversible inhibitor intermediate

3. suicide inhibitors medically important bc specific and become activated only when bound to enzyme (lower side effects)

1. is serine ionizable (acid/base reactions) under physiological conditions?

2. pH affect on enzyme activity

1. an AA reside that may participate in reaction catalyzed by pepsin (stomach protein is)

   1. serine pka 13 or Glu pka 3?

1. normally no bc of pka but it is in enzyme reaction

2. enzyme activity pH dependent

3. glu pka 3 close to where it operates at

define a and a’

1. a = inhibitor binding to free enzyme → EI complex

   1. competitive

2. a’ = inhibitor binding to ES complex → ESI

   1. uncompetitive or mixed

serine proteases:

1. what are they

2. what is the prototypical one

1. catalyze the hydrolytic cleavage of peptide bonds

2. chymotrypsin

chymotrypsin:

1. structure

2. catalytic triad?

3. substrate binding pocket?

4. highest level protein structure?

5. chymotrypsinogen?

1. 3 polypeptides (a, b, c chains) linked by disulfide bonds.

2. critical active site AA residues for catalytic reaction

   1. His Ser Asp

3. hydrophobic = binds (Trp/Phe/Tyr) to position substrate for active site

4. zymogen (proenzyme) form before cleavage makes it chymotripsin

   1. 
      

1. 
   

2. acylation then deacylation

chymotrypsin:

1. what does it do? 3 AAs?

2. what reaction does it catalyze

1. 2 step mechanism

1. cleaves other proteins AFTER large hydrophobic/aromatic side chains (Trp/Phe/Tyr)

2. catalyzes peptide hydrolysis

3. 2 steps

   1. acylation then deacylation

1. 3 steps how did scientists discover chymotrypsin had two step mech?

2. how did they identify active site residues

1. this protease cleaves peptide bonds but also slowly cleaves esters

2. product of ester cleavage reaction (p-nitrophenol) is yellow, used yellow pigment to track the reactions progress

3. saw a curve with both a pre-steady state burst phase in addition to a steady state phase, suggested it was a 2 step reaction

1. chemically labeled the specific residues in the active site

1. chymotrypsin 2 step mechanism?

2. what happens at each step?

3. what is water’s role?

1. fast step = acylation and release of p-NP

   1. yellow product fast

2. slow step = water hydrolysis of acyl-enzyme intermediate (deacylation)

   1. peptide bond breaking step

3. water directly attacks an acyl bond in an intermediate

   1. water doesn’t attack the peptide bond of the substrate

what is histidine’s role

catalyses 2 acid base reactions

how is chymotrypsin reaction is a great example of 3 things:

1. general acid-base catalysis

2. transition state stabilization

3. covalent catalysis

involved in 2 acid-base reactions

4 parts chymotrypsin active site:

1. triad

2. oxyanion hole

3. glysine -

4. hydrophobic pocket

1. what are the jobs of Asp102 and His57

1. triad = Asp102, His57, Ser195

2. oxyanion hole

3. glysine = helps w/ hydrogen bonding

   1. reaction cleaves peptide bond after aromatic group

4. hydrophobic pocket = where aromatic ring goes

1. to establish Ser195 as a strong nucleophile (O-)

chymotrypsin acylation phase:

1. starting products -(acylation)→ ending product

2. what happens (generally)

3. 4 steps

1. E + protein -(acylation)→ ES complex

2. covalent catalysis forms an ester linkage that forms acyl enzyme and the peptide bond is broken

3. 4 steps

   1. Substrate binds active site: forms ES complex

      1. His57 acts as general base and takes proton from Ser195 to establish Ser195 as a strong nucleophile (O-)

      2. His 57+ stabilized by Asp102 carboxylate

      3. H bonded chain makes Ser better n-phile

   2. Nucleophilic attack:

      1. Ser195-O- nucleophilic attack on C=O (covalent catalysis/ester linkage) to form short lived tetrahedral acyl-enzyme intermediate/transition state

      2. substrate has short lived O- charge stabilized by H bonding w/ Ser195 and Gly193

         1. H bond only occurs in this intermediate (TS stabilization)

   3. removal of LG:

      1. tetrahedral intermediate collapses reforming C=O and breaking peptide bond

      2. His57 acts as an acid protonates N terminus of LG peptide so it can leave

      3. product 1 (c-terminal half) is released

   4. End:

      1. acyl-enzyme remains (2nd transition state) = N terminal peptide is covalently bound to Ser195 via ester linkage

      2. C term of substrate diffuses away

acid-base catalysis of His57 in acylation phase v deacylation phase

1. acylation

   1. base to make See195 a good nucleophile (O-)

   2. acid to protonate N on the LG 1 (C term half of substrate)

1. deacylation

   1. base to make H20 good nucleophile (OH-0

   2. acid to protonate Ser195 to release LG2 (N term half of substrate)

1. ES complex v acyl-enzyme intermediate

2. Gly193 role?

3. deacylation or acylation phase?

1. ES complex = double bonded O H bonded to Ser195

2. acyl-enzyme intermediate/TS= single bonded/neg charge O- stabilized by H bonding w/ Ser195 and Gly193 in the oxyanion hole

3. TS stabilization

4. acylation phase

chymotrypsin deacylation phase:

1. starting products -(acylation)→ ending product

2. what happens (generally)

3. 4 steps

1. Acyl-enzyme -(hydrolysis)→ chymotrypsin

2. ester linkage of acyl-enzyme is hydrolyzed to regenerate original chymotrypsin

4 steps

5. Substrate binds:

   1. H20 enters active site

6. Nucleophilic attack:

   1. His57 acts as base and deporotonates H20

   2. OH- nucleophilic attack on C=O (covalent catalysis/ester linkage)

   3. oxyanion hole stabilizes short-lived tetrahedral acyl-enzyme intermediate

7. remove LG

   1. tetrahedra intermediate collapse

   2. His57 acts as an acid protonates Ser195 (-OH) facilitates displacement

   3. 2nd product (n-terminal half) diffuses

8. End:

   1. system reset, ready to accept new substrate

2 transition states

4 acid base reactions

2 covalent catalysis

differentiate b/t tetrahedral intermediate and acyl-enzyme intermediate

1. when substrate carbon has 4 bonds and covalently bound to enzyme

   1. When (O-) in oxyanion hole!!!

2. any time substrate or a substrate half is covalently bonded to enzyme

how are HIV proteases similar and 4 ways different than chymotrypsin?

1. similar

2. active site

3. water

4. complexes

5. cleave location

1. similar = cleaves proteases

2. active site = aspartate = aspartyl protease

3. water DOES directly attack peptide bond in HIV protease

4. no covalent enzyme-substrate complex

5. cleaves peptide bond b/t The and Pro AA pairs

HIV protease:

1. structure?

2. 3 step mechanism (difference w/ chymotrypsin)

1. homodimer (2 same proteins in quad structure), each with 2 Asp25s at the active site

2. 3 steps

   1. substate binding:

      1. hydrophobic pocket

   2. nucleophilic attack:

      1. General base catalysis: Asp25 (O-) takes H2O H+, OH- attacks carbonyl carbon

      2. tetrahedral intermediate (transition state) with C-O- stabilized by H bonding w/ Asp 25s

      3. (NO covalently attached intermediate, NO acyl-enzyme, NO oxyanion hole)

   3. remove LG:

      1. tetrahedral intermediate collapses to C=O (N term half released from 1 Asp25)

      2. other Asp25 acts as acid protonates C term half

How do Anti-HIV protease inhibitors work

1. they’re transition state analogs (mimic HIV protease’s tetrahedral transition state)

   1. OH mimics tetrahedral transition state O-

   2. benzyl gorup fits hydrophobic pocket

Enolase:

1. how does it catalyze its reaction

2. 3 parts active site

3. 2 parts reaction

1. divalent cations = uses 2 Mg2+ cofactors

2. 2 Mg2+, Lys345, Glu211

3. 2 steps

   1. Lys345 acts as general base and accepts substrate C2 proton

      1. C2 made more acidic by adjacent carboxyl (C=O)

      2. enolate intermediate C-O- charge stabilized by 2 Mg2+ (helps rearrange double bond)

   2. remove LG:

      1. Glu211 acts as general acid, protonates C-OH, and makes the -OH a better leaving group

      2. LG = H2O

1. what is antibiotic target?

2. specifically?

3. how does cross-linking happen?

   1. 2 step reaction mechanism

   2. transpeptidase?

4. why can we target peptidoglycan synthesis?

1. peptidoglycan synthesis

2. cell wall synthesis requires cross-linking of peptidoglycan molecules w/ each other

3. how

   1. peptidoglycan chains linked via D-ala containing peptidecross bridges

   2. transpeptidase carries out cross linking

4. cross linking mechanism

   1. transpeptidase active site (Ser-OH) attachés carbonyl of peptide bond of a D-ala

      1. cleaves a 2nd d-ala

      2. covalent linkage b/t substate, peptidoglycan, and transpeptidase

   2. peptide bond formation crosslinks 2 adjacent peptidoglycan chains (transpeptidase released)

      1. peptidoglycan chain #2 rather than water does 2nd nucleophilic attack

      2. transpeptidase = one peptide bond is replaced w/ another

5. because people have L-amino acids and D-ala is a D amino acid

B-lactam antibiotics:

1. what do they do

2. how

3. basic structure + how different

4. evolutionary arms race?

   1. step 1

   2. step 2

   3. step 3

1. inhibit transpeptidase via suicide inhibition = irreversible covalent inhibitor

2. has B-lactam ring that looks like a peptide bond and mimics d-ala

3. all have b-lactam ring, side chain, thiazolidine ring that mimic substrate. differ by r groups

4. antibiotic resistance

   1. penicillin

   2. penicillin resistance

      1. bacteria developed transpeptidase (B-lactamase) that breaks down penicillin so it can’t mimic (inactive transpeptide

   3. clavulanic acid

      1. suicide inhibitor of b-lactamase so it can’t inhibit penicilin

chymotrypsin 8 steps according to cummins

1. general base catalysis

   1. his57 makes ser195 nucleophile

2. covalent catalysis and TS stabilization (oxyanion hole)

   1. ser195 nucleophilic attack forms tetra O- in o hole w/ Gly

3. acid catalysis

   1. tetrahedral collapse and this57 protonates LG

4. product release

5. general base catalysis

   1. his57 makes water nucleophile

6. covalent catalysis and TS stabilization (oxyanion hole)

   1. OH- nucleophilic attack forms tetra O- in o hole w/ guy

7. general acid catalysis

   1. tetra collapse his57 protonates Ser195-OH

8. product release

1. allostery

   1. homo v heterotropic, where bind?

   2. positive or neg

2. covalent modification

3. cleavage

4. neg allosteric modulator v inhibitor?

1. reversible non-covalent binding of homotropic or heterotropic modulators (activator or inhibitor)

   1. homo = multisubunit enzyme that can bind more than one substrate at a time, but binding of one alters the binding of another

      1. often bind catalytic protein active site

   2. hetero = not a substrate, modulator binds somewhere else other than active site on enzyme but modifies active site activity

      1. modulator and substrate are DIFFERENT

      2. often binds regulatory subunits

2. like phosphorylation

3. activation of zymogens (pro-enzymes, not pro-protein) by proteolytic cleavage

4. inhibitor = no activity, neg also = reduced activity

1. 
   

1. allosteric enzymes show what relationship on V0 v. [S] plot?

2. why

3. how similar to normal non regulatory micaelis Minton?

4. how do positive and negative allosteric modulators affect the curve?

5. what do modulators change?

6. KEY characteristic of sigmoidal kinetics

1. allosteric enzymes = sigmoidal instead of normal hyperbolic curve

2. usually complex multimer proteins w/ complex reaction

3. still reach saturation at high [S]

4. positive = hyperbolic, shift left (high activity/r state)

5. negative = shifts right (low activity/T state)

6. there are many diff kinds that could change max or rate etc.

7. small changes in modulator concentration can have large affects on activity

aspartate transcarbamoylase (ATCase):

1. what does it do

2. how regulated (4) / how plot move

3. why sigmoidal

4. homo or hetero?

1. catalyses reaction of aspartate

2. has regulatory subunits and catalytic subunits

   1. catalytic bind substrate

   2. regulatory binding change conformation

      1. aspartate and carbamoyl phosphate = positive homotropic (substrate)

      2. ATP = positive heterotropic allosteric modulator → shift left relaxed state / high affinity

      3. CTP = neg heterotropic allosteric modulator → shift right tense state / low affinity

3. binding of substrates aspartate and carbamoyl phosphate = transition from T state →R state

1. ATCase both homo and hetero allosteric kinetic behavior

aspartate transcarbamoylase (ATCase):

1. why does ATP activate and CTP inhibit?

2. what’s the mechanism?

3. what are pathways? what’s usually the final product usually do?

4. rationale behind ATP activating and CTP inhibiting?

1. allosteric control of ATCase by CTP = feedback inhibition

2. CTP is product produced at end of long pathway and acts feedback inhibitor of earlier ATCase reaction to control level of CTP in the cell

3. processes where product of one enzyme becomes substrate of next each step catalyzed by a different enzyme

   1. final product usually heterotropic allosteric inhibitor of earlier enzymes in the pathway

4. rationale

   1. ATP activates = want to make stuff when have energy

   2. CTP feedback inhibitor = don’t want to waste E making too much

1. how covalent modification regulate enzyme function

2. how permanent are covalent mods

1. many different types/affects of cov mods catalyzed by enzymes

2. permanent until removed by another enzyme

1. describe usefulness of phosphorylation in enzymatic regulation and how glycogen phosphorylase is regulated in response to phosphorylation

Phosphorylation:

1. catalyzed by

   1. what do they reconize

2. removed by

   1. specificity

3. how is glycogen phosphorylase regulated by phosphorylation?

1. catalyzed by kinases

   1. specific, kinases recognize seq motifs

2. removed phosphatase

   1. not specific

3. its really complex, but simple version:

   1. glycogen phosphorylase switches b/t active a form and inactive b form by phosphorylating 2 Ser residues

      1. phosphorylase kinase activates = turns b → a

      2. protein phosphatase 1 inhibits = turns a → b